Joris van Dongen

92 Chapter 4 MATERIAL & METHODS Liposuction and FAT procedures Liposuction as well as the FATprocedure were executed as described in the study of van Dongen et al. between September 2017 – 2018. 18-20 All included patients gave informed consent according to the Declaration of Helsinki. In short, adipose tissue harvesting was performed with a Sorenson lipoharvesting cannula (Tulip, Medical Products, San Diego, CA) during normal liposuction procedures in three patients. Lipoaspirate was centrifuged after decantation and mechanically dissociated either by the three-hole fractionator or the one-hole fractionator (Tulip, Medical Products, San Diego, CA, fig. 1). After mechanical dissociation, samples from both types of fractionators were centrifuged again yielding a comparable amount of oil (8.4 ml ± 0.4), tSVF (1.1 ml ± 0.4) and infiltration fluid fraction containing a pellet (0.5 ml). One sample of centrifuged lipoaspirate of each patient (n=3) was used as a control. Immunohistochemistry & Masson’s Trichrome Samples of tSVF obtained by the one-hole fractionator and control lipoaspirate (n=3) were fixed in 10% formalin in phosphate buffered saline (PBS), embedded in paraffin and 4 µm sections were cut. Immunohistochemistry i.e. α -Smooth Muscle Actin (SMA) and Perilipin, as well as Masson’s Trichrome staining were performed according to the protocol of van Dongen et al . 16 Primary antibodies used in this study were α -SMA (1:200, Abcam, Cambridge, UK) and Perilipin (1:200, Abcam, Cambridge, UK). Secondary antibodies used were polyclonal Rabbit anti-Mouse for α -SMA (1:100, DAKO, Glostrup, Denmark) as well as polyclonal Goat anti-Rabbit for Perilipin (1:100, DAKO, Glostrup, Denmark). A tertiary antibody was only used for α -SMA (polyclonal Swine anti-Rabbit for 1:100, DAKO, Glostrup, Denmark). All samples were visualized and evaluated by light microscopy (Leica Microsystems, DM IL). Cell Isolation and Culture Samples of tSVF obtained by the one-hole fractionator and the three-hole fractionator as well as control lipoaspirate (n=3) were enzymatically dissociated in 0.1% collagenase A, 1% bovine serum albumin in PBS and cultured according to our previously published protocol. 16 Cells were counted upon staining with trypan-blue in a Bürker Türk counting chamber. Flow cytometry Cells collectedfromenzymaticallydissociatedtSVFsamples i.e. theone-hole fractionator and the three-hole fractionator (n=3) were analyzed for surface marker expression